Nucleosides. 5. Synthesis of guanine and formycin B derivatives as

Nucleosides. 5. Synthesis of guanine and formycin B derivatives as potential inhibitors of purine nucleoside phosphorylase. Ji Wang Chern, Horng Yuh L...
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J . Med. Chem. 1993,36, 1024-1031

1024

Nucleosides. 5.’ Synthesis of Guanine and Formycin B Derivatives as Potential Inhibitors of Purine Nucleoside Phosphorylase Ji-Wang Chern,’ Horng-Yuh Lee, and Chien-Shu Chen Medical Laboratories and Institute of Pharmacy, National Defense Medical Center, P. 0. Box 90048-512, Taipei, Taiwan, ROC(100)

Donna S. Shewach,+ Peter E. Daddona,$ and Leroy B. Townsend’ Departments of Pharmacology and Biological Chemistry, Medical School, Department of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry, T h e University o f Michigan, Ann Arbor, Michigan 48109-1065 Received August 19, 1992

In an effort to develop potent human purine nucleoside phosphorylase (PNP) inhibitors as immunosuppressive and chemotherapeutic agents, several 8-aminoguanine derivatives were synthesized and evaluated as potential P N P inhibitors. These studies were designed to investigate the hydrophobic effect of a substituent on the N-9 of the purine heterocycle and/or the C-5’ positions. Compounds such as 8-aminoguanosine, guanosine, formycin B, and 8-aminoacyclovir containing a p(fluorosulfony1)benzoylmoiety were synthesized. The affinity of these compounds to erythrocytic P N P was determined and none of these compounds showed a better affinity than those of the parent compounds. However, we found that the effect of hydrophobicity a t the N-9 and the C-5’ positions might play an important role in binding to the active site of PNP. Thus, 8-amino-5’-deoxy-5’-(phenylthio)guanosine (19) was found to be the best inhibitor in this series of compounds with a Ki = 0.45pM.

Introduction Purine nucleosidephosphorylase (PNP; orthophosphate ribosyltransferase; EC 2.4.2.W is considered an essential enzyme in the purine salvage pathway, catalyzing the phosphorolysis of guanosine, inosine and their 2’-deoxyribonucleoside derivatives to the respective purine bases. The discoveryof T-cell-related immunodeficiency diseases associatedwith a lack of PNP3v4has prompted considerable interest in developing inhibitors of this enzyme as a selective approach to cancer ~hemotherapy.~ It is welldocumented that in a PNP-related immunodeficiency state, 2’-deoxyguanosine accumulates and this nucleoside is phosphorylated preferentially by immature T cells to the toxic 5’4riphosphate derivative, which results in inhibition of DNA synthesis.6g It has recently been reportedlo that human PNP deficiency was caused by a single base change in the DNA genome.1° T-cell lymphopenia is associated with a PNP-deficient patient; therefore, it was suggested that a PNP inhibitor might be used to treat T-cell leukemias, to counter autoimmune diseases without destroying the patient’s humoral immunity and to treat tissue rejection after organ transplantati0n.l’ Furthermore, PNP inhibitors might provide some potential use for antimalarial treatment because the intraerythrocytic malaria parasite requires hypoxanthine and guanine formed by PNP as a source of nucleic acid synthesis and energy metabolism.12 The importance of PNP inhibitory activity for lymphocyte development and function in chemotherapy has generated considerable interest in a systematic examination of the structure-activity relationships of substrates for human erythrocytic PNP.13J4 To date, several inhibitors of PNP have been identified and the majority of these compounds resemble purine bases or nucleo~ i d e s . ~ JIt~ has J ~ been established that halogen at the +

Department of Pharmacology.

t Department of Biological Chemistry.

C-5’ position of specific PNP substrates results in improved affinities but greatly decreased substrate activity, for example, 5’-deoxy-5’-iodoformycin(Ki = 7 pM)illustrated that the binding to PNP by iodination at C-5’ of the parent compound17 increased the activity by 10-fold. X-ray crystallography revealed that the 5’-iOdO atom is juxtaposed over the phenylalanine residue of the active site of PNP, indicative of the enhancement of binding to PNP probably due to the charge-charge interaction of the halogen atom with the phenylalanine residue in the active site of PNP.ls In a previous communication, we assumed that 8-aminoguanine plays an important role in recognizing and binding to the active site of PNP, and through an attempt to investigate the bulky tolerance at the N-9 position of 8-aminoguanine, we found that 8-amino-9benzylguanine (8-ABG)is apotent inhibitor of both human

erythrocyte and parasite PNP.12J9 The intact cell experiments showed that 8-ABG acted as a competitive (with inosine) inhibitor with a Ki value 0.22 p M and could potentiate the toxicity of 2’-deoxyguanosine to MOLT-4 T-lymphoblasts in culture. We reasoned that the increased affinity of 8-ABG to PNP, more than 10-fold by a benzyl group on the N-9 position of 8-aminoguanine, is probably due to this hydrophobic interaction between the benzyl group and the phenylalanine residue (Phe200)of the active site of PNP. Such a hydrophobic region adjacent to the active site of an enzyme has been observed on dihydrofolic reductase,20guanasePl and thymidine phosphorylaseP2 and the exploitation of the hydrophobic regions in the active site for the design of reversible and irreversible inhibitors have been previously studied.

0022-2623/93/1836-1024$04.00/00 1993 American Chemical Society

Potential Inhibitors of Purine Nucleoside Phosphorylase

Journal of Medicinal Chemistry, 1993, Vol. 36, No. 8 1025

Scheme I1 a

Scheme I a .NHC-OCH,

3. R-NHP. RLH

2

4, R.NHR~,

RLNO

5, R.NHP, R'.NH~

0

0

,/ RZ-

vi

8

a: .(CH~)YCH~ b .(CHds-CM)H

' D HOH&

(i) Amines, ethanol; (ii) NaN02; (iii) dithionite; (iv) SCNC(=O)OCH3, CH3CN; (v) DCC, DMF, room temperature; (vi) KzC03, MeOH, reflux; (vii) 10% aqueous NaOH.

Accordingly, we selected 8-aminoguanine and the corresponding nucleoside as our "lead compounds" for studies to investigate the hydrophobic effect of substituents on the N-9 of the purine heterocycle and C-5' positions of the carbohydrate moiety.

Results and Discussion Chemistry. 8-Amino-9-substituted-guanines(9a-e) were synthesized (Scheme I) by a method previously published23from our laboratory. This involves the initial synthesis of the key intermediates, 2-amino-4-(substitutedamino)pyrimidin-6-ones (3a-e) which were subjected to nitrosation with sodium nitrite and subsequent reduction with sodium dithionite to give the 2,5-diamino-4-(substituted-amino)pyrimidin-6-ones(5a-e). Compounds 5a-e were readily oxidized in the air; therefore, they were condensed with methoxycarbonyl isothiocyanate at once without further purification. Then cyclodesulfurization and ring annulation of the resulting thiourea derivatives 6-e with dicyclohexylcarbodiimide, followed by a ring transformation of the oxazolo[5,4-d]pyrimidines7a-e led to the formation of the imidazo[4,5-dlpyrimidines8a-e. Subsequent hydrolysis of 8a-e with a 10% sodium hydroxide solution afforded the target compounds. In the 8-aminoacyclovir derivatives series (Scheme 111, 8-aminoacyclovir (15) was synthesized by a previous method.24 Bromination of acyclovir (13)was effected using saturated bromine water to give 8-bromoacyclovir (14) in 84% yield. Treatment of the bromo compound with 70% aqueous hydrazine a t reflux afforded compound 15. The iodination of acyclovir and 8-aminoacyclovir, with triphenylphosphine and iodine in N,N-dimethylformamide using Moffat's pr0cedure,~5furnished 9-[ (2-iodoethoxy)methyllguanine (16) and 8-amino-9-[(2-iodoethoxy)methyllguanine (171, respectively. 8-Amino-5'-deoxy-5'-(phenylthio)guanosine (19) was prepared (Scheme 111)in 59 7% yield by a simple treatment of 8-aminoguanosine ( 1 2 P with diphenyl disulfide in the presence of tributylphosphine.27 5'-[p-(Fluorosulfonyl)benzoyllguanosine (21)28was previously prepared for a ligand study by a direct acylation of the 5'-OH group of guanosine hydrochloride with p-(fluorosulfony1)benzoyl chloride in HMPA. For our studies, 21 was synthesized by a modification of this

18

17

(i) (c6H5)3PII2,DMF, room temperature, 39%; (ii) Brz, H20; (iii) p(fluorosulfony1)benzoyl chloride, HMPA, -10 "C, 82%; (iv) 50% hydrazine, 48 h; (v) (C6H&P, DMF, 12, room temperature, 23 % . (I

Scheme I11 a 0

11. R-Br

10

19

12, R-NHz

HO

dH

20, R-NHz 21, R-H

(i) Bromine water, room temperature; (ii) 50% hydrazine, reflux; (iii) diphenyl disulfide, (CsH&P, pyridine, room temperature, 60 h, 59 % ;(iv) p(fluorosulfony1)benzoylchloride, HMPA, room temperature 37%.

procedure by a direct acylation of the 5'-OH of guanosine with p(fluorosulfony1)benzoyl chloride, instead of the hydrochloride salt. The structure of this compound was determined on the basis of its 'H-NMR spectrum. However, the treatment of 8-aminoguanosine with p(fluorosulfony1)benzoyl chloride in HMPA resulted in a nonselective acylation. The treatment of 8-aminoguanosine dihydrochloride with p(fluorosulfony1)benzoyl chloride in HMPA afforded the dihydrochloride salt of the desired compound. The neutralization of 8-amino5'- [ p -(fluorosulfonyl)benzoyl] guanosine dihydrochloride with triethylamine in ethanol was unsuccessful. The fluorosulfonyl moiety was found to be very unstable in aqueous basic solution. However, treatment of this salt with triethylamine in anhydrous DMF furnished 8-amino5'- [@-fluorosulfonyl)benzoyllguanosine (20) as established by 'H-NMR spectroscopy and elemental analysis. The structurally related compounds, 9-1[2-[p-(fluorosulfonyl)-

1026 J o u r n a l of Medicinal Chemistry, 1993, Vol. 36, No. 8

Scheme IV a

““w N

HO

-

5 H L - - N

N

D o

OH

OH

HO

22

23

24

(i) (Cp,H&P, diphenyl disulfide, pyridine, room temperature, 89 % ; (ii) p(fluorosulfony1)benzoyl chloride, HMPA, room temperature, 12 h, 71%.

Table I. Kinetic Constants for Human P N P compounds 8-aminoguanine 8-aminoguanosine (12) 8-amino-9-benzylguanine (1) 8-amino-9-hexylguanine (9a)

8-amino-9-(carboxypentyl)guanine (9b) 8-amino-9-(cyclohexylmethyl)guanine (9c) d-amino-9-(naphthylmethyl)guanine(9d) 8-amino-9- [m-(hydroxymethyl)phenyllguanine (9e) acyclovir (13) 8-bromoacyclovir (14) 8-aminoacyclovir (15) 9- [ (2-iodoethoxy)methyllguanine(16) 8-amino-9-[ (2-iodoethoxy)methyllguanine(17) 8-amino-5’-deoxy-5’-(phenylthio)guanosine (19) 5’- [p-(fluorosulfonyl)benzoyllguanosine(21) 8-amino-5’-[p-(fluorosulfonyl)benzoyllguanosine(20) 9- [ [2-[ p -(fluorosulfonyl)benzoyl] ethoxyl methyl1guanine (18) 5’-[p-(fluorosulfonyl)benzoyllformycinB (24) ~~

Ki (wM) 0.8 7.0

0.2 12.4 10.0 a

a b 180.0 18.7

13.4 15.0

23.0 0.45 93.0

37.0 C

>300 26.0 5’-deoxy-5’-(phenylthio)formycinB (23) 0 Insoluble. b No inhibition a t 1 mM. 95% inhibition a t 1 mM. ~

benzoyl]ethoxy]methyllguanine (18) and 5’- [p-(fluorosulfonyl)benzoyllformycin B (24)were synthesized in 82 and 71 5% yields, respectively. 5’-Deoxy-5’-(phenylthio)formycin B (23)was obtained in 89% from formycin B (22)by the same approach as that used for the synthesis of 19.

Biological Aspects PNP activity was assayed in dialyzed extracts from MOLT-4 T-lymphoblasts or human erythrocytes using a previously described radiochemical assay.ll Kinetic constants were estimated from double reciprocalplots of initial velocity versus substrate (inosine) concentration. All inhibitors were competitive with inosine. The results are summarized in Table I. Several aspects of the stereoelectronic requirements of substrates for human erythrocytic PNP have been studied15J6and it has been demonstrated that groups on the such as NHCH3 and N(CH3)z C-8position larger than “2, can not be accommodated by PNP. Also, purine analogues with OH, SCHs, and propyl groups substituted a t C-8 show little or no PNP inhibitory activity.29 In a previous

Chern e t al.

communication, we assumed that an electron-donating group at the C-8 position, without producing excessive steric hindrance, would increase the electron density at N-7 and thus increase the affinity of the compound toward the enzyme. On the basis of this assumption, we found 8-amino-9-benzylguanine was a potent PNP inhibitor and concluded that the length of the hydrophobic substituent cannot exceed a benzyl group.1g In order to investigate the hydrophobic character on both sides of the benzyl were group, 9-cyclohexyl-,and 9-naphthyl-8-aminoguanine synthesized and evaluated. Unfortunately, due to the poor solubility of these compounds, the data on their PNP inhibitory activity were not obtained. The PNP inhibitory activity of compounds 13-17 and 9-hexyl-8-aminoguanine are not better than those obtained for 9c-e. However, we found that by adding an amino group at the 8 position of acyclovir, to obtain compound 15 led to an increase of more than a 10-fold affinity to PNP. A recent report on a metabolite of the antiherpetic drug acyclovir, acycolvir diphosphate, revealed this compound was the most potent inhibitor of PNP to date with a Ki = 0.0087 pM (determined at a low inorganic phosphate con~entration.~~) The Ki value increased to 0.51 pM when the phosphate concentration was increased to 50 pM, suggesting that there is a positive charge adjacent to the active site which is binding to an anionic charge on the terminal phosphate ester moiety of acyclovir diphosphate. Accordingly, metabolically stable “multisubstrate” acyclic nucleotide analogues containing a purine and phosphatelike moiety such as 9-(phosphonoalky1)hypoxanthinesand guanines have been synthesized and evaluated as potential purine nucleoside phosphorylase inhibit0rs.3~-33In this series, 9-(5-phosphonopentyl)guaninewas considered as the most potent with a Ki = 170 nM. 8-Amino-9(carboxypenty1)guanine (9b),containing a carboxylate ion at the terminal end, which is considered as an isostere of phosphono or phosphate, is a multisubstrate analogue. However, cpmpound 9b demonstrated a weak PNP inhibitory activity with a Ki = 10.0 pM. Modifications at (2-5’ of the ribose moiety in a purine nucleoside is considered to be a promising approach to develop PNP inhibitors. It is generally agreed that a halogen atom at the C-5’ position provides a better affinity for the nucleoside to PNP.12J6J7 Similarly, the PNP inhibitory activity of acyclovir was increased 10-fold by replacing the 2-hydroxy group with an iodo atom to give 9-[(2-iodoethoxy)methyl]guanine(16). However, a similar replacement of the 2-hydroxy group of 8-aminoacyclovir with an iodo atom did not enhance the affinity to PNP. We found that compound 19,having a phenylthio group at the (2-5’ position, with a Ki = 0.45 pM was 40-fold better than that of 8-aminoguanosine and possessed potency equal to 8-amino-5’-deoxy-5’-chloroguanosine.12 However, compound 23, with the same substituent at the (2-5’ position of formycin B, showed poor inhibitory activity with a Ki = 26 pM which is weaker than that of 19. Although compound 23 is not as potent as 5/-deoxy-5‘iodoformycin (Ki = 7.0 pM), it is still better than the parent compound formycin B (Ki = 100pM). The results obtained from these studies coincide with reports that halogens or a thiomethyl group at the C-5’ position of specific PNP substrates resulted in unchanged or improved affinities but greatly decreased substrate activity.17 The increased to affinity of 8-amino-5’-deoxy-5’-(phenylthio)guanosine PNP was probably due to an interaction of the 5’-

Potential Inhibitors of Purine Nucleoside Phosphorylase

Journal of Medicinal Chemistry, 1993, Vol. 36,No. 8 1021 0

compound

Figure 1. Superimposition of compound 19 and 23 in energyminimized conformation state.

phenylthio group with the phenylalanine residue and is indicative of a hydrophobic region adjacent to the active site of PNP. The active site of purine nucleoside phosphorylase has been recently characterized using X-ray diffraction data.lk This study revealed that guanine, hypoxanthine, and certain analogues donate a hydrogen bond at N-1 and accept a hydrogen bond at 0-6. Am-243 and Lys-244 are near 0-6 while Glu-201is near N-1. Futhermore,the amino group at C-2 of guanine is in position to form an additional hydrogen bondwithGlu-201. According to this description of the purine binding site of PNP, we assumed that the 8-aminoguanine moiety and the pyrazolopyrimidme moiety of formycin B could enter the active site of PNP with the ribose moiety being outside of the binding site. On the basis of this assumption, Figure 1 shows energyminimized conformations of 19 and 23 (superimposed), generated using the ALCHEMY 11 molecular modeling program.18b These 8-aminoguaninederivatives and formycin B analogues provide the sane arrangement of hydrogen bond donors and receptors in the active site of PNP while the riboee and the 5’-phenylthio group of 19 and 23 are distorted in different directions. However, the 5’-phenylthio moiety of 19 is anti to the 8-amino group while the 5’-phenylthio group of 23 is syn to the 8-position. The latter conformation is very similar to that reported for 5’-iodoformycin B binding to the active site of PNP (in the X-ray diffraction data). The introduction of a phenylthio group at the 5’ position leads to a 40-fold improvement in the 8-aminoguanasineseries, but only a 4-fold improvement in the formycin B series, and it is probably due to the phenylthio groupsselecting a different hydrophobic site. It should be noted that one side of the ‘ribose pocket” has been characterized as hydrophobic and includes at least 4 aromatic residues (Phe-200, Tyr88, and His-257 from one subunit, along with Phe-159 from the neighboring subunit). Accordingly, we propose that two hydrophobic binding sites might exist in the pocket as illustrated in Figure 2. The hydrophobic area A is in or near the active site and plays a major role while theotherareaisintheneighboringsubunitandmostlikely plays a minor role.

Figure 2. Proposed three binding sites for purine nucleoside phosphorylase inhibitors. Shaded part is purine binding site, A and B represent two hydrophobic areas.

A t the outset, we presumed that the Eaminoguanine nucleus might be responsible for the recognition and binding of 8-aminoguanosine to the active site of PNP. This prompted us to make the assumption that perhaps there is another pocket for the C-5’ position of the ribose whichwould allow us to use achemically reactive functional group such asp-(fluorosulfony1)bemoylgroup on the C-5’ position of the ribose for irreversible binding to PNF’. However, nucleosides containing this sulfonyl fluoride moiety have been previously synthesized and evaluated as affinity labeling agents for adenosine nucleotide sites in glutamate dehydr~genase?~ pyruvate kinase,% RNA and the mitopolymerase,36 phosphofr~ctokinase,3~~~~ chondrialF1-ATPase.” Weexpededthis sulfonylfluoride moiety to be capable of functioning as an electrophilic functional group in covalent reactions with several classes of aminoacidssuchas lysine, tyrosine, histidine, and serine in the active site of PNP.40 Thus, the synthesis of 8-aminoguanosine with several chemically reactive functions attached to the (2-5’ position became one of our primary goals in the design of PNP inhibitors, e.g., 9-[[2[p-(fluorosulfonyl)henzoyllethoxylmethyllguanine(18). 8-amino-5’- [p-(fluorosulfonyl)benzoyllguanoaine(20), 5’[p-(fluorosulfonyl)benzoyllguanosine(Zl), and 5’-[p-(fluorosulfonyl)benzoyllformycinB(24) were synthesizedand evaluated. As can be seen from Table I, none of these compoundsshowed irreversible inhibitory activity to PNP. In fact, the inhibitory activities of these compounds are even weaker than those of the parent compounds. This might be because there is no nucleophilic residue at the active site of PNP and/or the sulfonyl fluoride moiety is too large to fit into the active site of PNP. However, the enzymaticinhibitoryactivityofcompound20isstillbetter than that of 21, indicative of the importance of having an amino group at the &position. In summation, the influence of an amino substituent a t the 8-position of guanosine was consistent with that of the previous assumption that &aminoguanine plays an important role in recognition and binding to the active site of purine nucleoside phosphorylase. The area around the 9-pasition of8-aminoguanineismost likely apocket which can accomodate a larger group and could provide a labeled and chemically reactive functional group. The same phenomena was also observed at the C-5‘ position of the ribose moiety of 8-aminoguanosine. However, achemically reactive functional group a t the C-5’ position did not

1028 Journal of Medicinal Chemistry, 1993, Vol. 36, No. 8

provide an irreversible inhibitor and would suggest that nucleophilic residues (if they are in that area) are not in the proper juxtaposition for a reaction to occur. Experimental Section Melting points were obtained on a Electrothermal apparatus and are uncorrected. Infrared spectra were recorded on a PerkinElmer Model 983 G spectrophotometer; 'H and 13C nuclear magnetic resonance spectra were recorded on a Bruker Model AM-300 spectrometer using Me2So-d~as the solvent and internal standard. UV spectra were recorded on a Shimadzu UV-210 A spectrophotometer. AICA hydrochlorideand AICA-ribosidewere purchased from Sigma Chemical Co. Elemental analysis were carried out in Cheng-Kung University, Tainan, Taiwan. 2-Amino-4-(n-hexylamino)pyrimidin-6-one (3a). A mix(2)(5.5 g, 37.67 mmol) ture of 2-amino-4-chloropyrimidin-6-one and n-hexylamine (15 mL, 0.11 mol) in anhydrous ethanol (100 mL) was heated at reflux in an oil bath for 2 days. The mixture was evaporated in vacuo (water pump) at 70 "C to obtain an oily residue. A solid was formed by the addition of methanol (50 mL) to this oily residue. The solid was collected by filtration and recrystallized from methanol to give 5.1 g (71%)of 3a: mp 179180 OC; 'H NMR (270 MHz, DMSO-dg): 6 0.85 (t,3 H, CH3),1.24 (8, 4 H, 2CH2), 1.43 (m, 2 H, CHz), 3.00 (m, 2 H, CHz), 4.38 (s, 1 H, =CH), 6.08 (s, 2 H, "2, D20 exchangeable), 6.30 (br s, 1 H, NH, D20 exhangeable), 9.63 (s, 1H, NH, DzO exchangeable). Anal. (C10H18N40). C, H, N. 2-Amino-4-[ (cyclohexylmethyl)amino]pyrimidin-6-one (3c). Compound 3c was prepared in 45 % yield using a procedure similar to that which afforded 3a. An analytical sample was prepared by recrystallization from a mixture of ethanol and water: mp 226 "C dec; lH NMR (100 MHz, DMSO-&) 6 0.781.75 (m, 11 H), 2.87 (t, 2 H, CHz), 4.40 (9, 1H, =CHI, 6.07 (s, 2 H, "2, D20 exchangeable), 6.35 (t, 1H, NH, D20 exchangeable), 9.60 (s, 1H, NH, D 2 0exchangeable). Anal. (CllH18N40) C, H, N. 2-Amino-4-[(naphthylmethyl)amino]pyrimidin-6-one(3d). Compound 3d was prepared in 34% yield using a procedure similar to that which afforded 3a: mp 260 "C dec; 'H NMR (100 MHz, DMSO-dg) 6 4.48 (9, 1 H, =CH), 4.76 (d, 2 H, CHz), 6.17 (s, 2 H, NH2, D20 exchangeable), 6.93 (t, 1 H, NH, D20 exchangeable), 7.42-8.15 (m, 7 H, ArH), 9.64 (br s, 1H, NH, D 2 0 exchangeable). Anal. ( C ~ ~ H I ~ N ~ O J C, / ~ H, H ~N.O ) 2-Amino-4-( a-hexylamino)-5-[ 1-[ 3-(methoxycarbony1)thioureido]]pyrimidin-6-one(6a). Sodium nitrite (4.0 g, 57.97 mmol) in water (10mL) was added to a mixture of 3a (4.0 g, 19.02 mmol),glacialaceticacid (5mL),andwater (150mL) withstirring a t room temperature. After 12 h, the red solid was collected by filtration and washed with water (50 mL). The red solid was then suspended in water (150 mL) and heated on a hot plate with stirring. Sodium dithionite was added to the hot stirring mixture in small portions over a period of 1hour until the red color changed to a light yellow color. The solid was collected by filtration and was subsequently mixed with 3 equiv of methoxycarbonyl isothiocyanate in acetonitrile (100 mL). The mixture was heated at reflux for 5 h. After the mixture was cooled to room temperature, the solid was collected by filtration and washed with ether (50 mL). The solid was recrystallized from a mixture of DMF and water to afford 4.21 g (65%)of 6a: mp 224-225 "C. Anal. (C13H&03) C, H, N. 2-Amino-4-[ (carboxypenty1)aminol-5-[ 1-[3-(methoxycarbonyl)thioureido]]pyrimidin-6-one (6b). A mixture of 2-amino-4-chloroppimidin-6-one (1.5 g, 10mmol) and 6-aminohexanoic acid (1.5mL l2mmol) inanhydrousethanol (100mL) was heated at reflux in an oil bath for 2 days. The mixture was evaporated in vacuo (water pump) at 70 "C to obtain 3b (1.9 g, 7.92 mmol) as an oily residue. Sodium nitrite (1.6 g, 23.2 mmol) in water (5 mL) was subsequently added to a mixture of this oily residue (3b,1.9 g, 7.92 mmol) in glacial acetic acid (3 mL) and water (50 mL). After stirring at room temperature for 5 h, the red solid was collected by filtration and washed with water (100 mL). The solid was suspended in water (200 mL) and heated on a hot plate with stirring. Sodium dithionite was added to the hot stirring mixture in small portions over a period of 1hour until the color

Chern et al. had changed to a light yellow color. After the mixture was cooled to room temperature, and the solid was collected by filtration and mixed with 3 equiv of methoxycarbonyl isothiocyanate in acetonitrile (50 mL). The mixture was heated a t reflux in an oil bath for 6 h. The white solid was collected by filtration and recrystallized from a mixture of ethanol and water to give 1.78 g (60% from 3b): mp 221-222 "C;'H NMR (270 MHz, DMSOdg) 6 1.26 (9, 2H, CH2), 1.5-1.3 (m, 4H,CHz), 2.17 (t, 2H, CH2), 3.19 (4, ZH, CH2), 3.70 (8, 3H, CH3), 6.17 (t, lH, NH, DzO exchangeable), 6.27 (e, 2H, "2, D2O exchangeable), 9.93 (br s, lH, NH, D20 exchangeable),10.07 (s, lH, NH,DzOexchangeable), 11.11 (s, lH, NH, D20 exchangeable), 11.95 (9, lH, NH, D20 exchangeable). Anal. (C&oN60&3). C, H, N. 2-Amino-4-[ (cyclohexylmethyl)amino]-5-[1-[3-(methoxycarbonyl)thioureido]]pyrimidin-6-one (6c). Compound 6c was prepared in 79% yield using a procedure similar to that which afforded 6a: mp 222-224 "C; 'H NMR (100 MHz, DMSOdg) 6 0.86-1.78 (m, 11 H), 3.08 (t, 2 H, CHz), 3.71 ( 8 , 3 H, CH3), D20 exchangeable), 9.91 (8, 6.13 (t, 1H, NH), 6.26 (9, 2 H, "2, 1 H, NH, D20exchangeable), 10.08 (s, 1H, NH, D20 exchangeable),11.08 (s, 1H,NH,DzO exchangeable). Anal. (C14H22N&S) C, H, N. 2-Amino-4-[ (naphthylmethy1)aminol-5-[ 1-[3-(methoxycarbonyl)thioureido]]pyrimidin-6-one(6d). Compound 6d was prepared in 58% yield using a procedure similar to that which afforded 6a: mp 233 OC dec; 'H NMR (100 MHz, DMSOdg) 6 3.70 ( ~ , H, 3 CH3), 4.97 (d, 2 H, CHz), 6.29 ( ~ , H, 2 NHz, D20 exchangeable), 6.9 (t,1H, NH, DzO exchangeable), 7.34-8.14 (m, 7 H, ArH), 10.01 (s, 1 H, NH, D20 exchangeable), 10.18 (s, 1 H, NH, D20 exchangeable), 11.12 (9, 1H, NH, D20 exchangeable). Anal. (Cl~Hl~Ns03S-1/4H20) C, H, N. 2-Amino-4-[ [m-(hydroxymethyl)phenyl]amino]-5-[ 1-[3(methoxycarbonyl)thioureido]]pyrimidin-6-one(b). Compound 6e was prepared in 42% yield using a procedure similar to that which afforded 6a: mp 239-240 "C; 'H NMR (100 MHz, DMSO-d6)6 3.72 (s, 3 H, CH3), 4.44 (s, 2 H, CHz), 5.10 (br s, 1 H, OH, D20 exchangeable),6.43 (s,2 H, "2, DzO exchangeable), 6.90 (d, 1 H, ArH), 7.16 (t, 1 H, ArH), 7.37 (s, 1 H, ArH), 7.50 (d, 1 H, ArH), 8.02 (9, 1 H, NH, D20 exchangeable), 10.35 (8, 1 H, NH, D20 exchangeable),11.17 (s,l H, NH,DzO exchangeable). Anal. (C14H16N60&b1/2H20)C, H, N. 8-Amino-9-hexylguanine (9a). A mixture of 7a (1.35g, 4.38 mmol) and anhydrous potassium carbonate (1.31 g, 9.49 mmol) in anhydrous methanol (30 mL) was heated at reflux in an oil bath. After 6 h, the solvent was removed by evaporation in vacuo (water pump) at 70 "C. Water (100mL) was added to the residue, and the mixture was then heated at reflux in an oil bath for 24 h. The mixture was cooled to room temperature and the pH was adjusted to 5 with glacial acetic acid. The solid was collected by filtration and washed with water (50 mL). The crude product was recrystallized from 1N sodium hydroxide solution and glacial acetic acid to afford 0.82 g (75% ) of 9a: mp 300 "C; UV,,A nm (c X lo4) (MeOH-DMF, v:v, 8:2) 259 (1.5), 293 (0.9); (pH 1)289 (1.0); (pH 11) 255 (1.7); 'H NMR (270 MHz, DMSO-ds) 6 0.83 (t, 3 H, CH3), 1.23 (s, 4 H, 2 CHZ),1.55 (m, 2 H, CHz), 3.73 (t, 2 H, CHz), 5.83 (s, 2 H, "2, DzO exchangeable), 6.17 (s, 2 H, "2, DzO exchangeable), 10.44 (s, 1H, NH, D20 exchangeable). Anal. (C11H&O) C, H, N. 8-Amino-9-(carboxypentyl)guanine(9b). Dicyclohexylcarbodiimide (1.1g, 5.34 mmol) was added to a mixture of 6b (1.0 g, 2.69 mmol) and methanol (50 mL). The mixture was heated at reflux in an oil bath for 6 h. The solvent was then removed byevaporation to dryness in vacuo (water pump) at 70 "C. Boiling toluene (50 mL) was added to the residue, and the white solid was collected and washed with boiling toluene (50 mL). A mixture of this crude product and potassium carbonate (0.74g,5.34mmol) in methanol (50 mL) was heated a t reflux in an oil bath for 6 h. The solvent was then removed by evaporation in vacuo (water pump) at 70 "C. Water (50 mL) was added to the residue, and the mixture was heated at reflux for 12 h. The mixture was cooled to room temperature and the pH was adjusted to 5 with glacial acetic acid. The solid was collected by filtration and washed with water (100 mL). The crude product was recrystallized from 1 N sodium hydroxide solution and glacial acetic acid to afford 0.51 g (65% ) of 9b: mp 300 "C; 'HNMR (270 MHz, DMSO-ds)6 1.30-1.24 (m, 2 H, CHz), 1.88-1.4 (m,4H, CH2),2.18

Potential Inhibitors of Purine Nucleoside Phosphorylase

(t, 2 H, CHz), 3.74 (t, 2 H, CHz), 5.99 (5, 2 H, NHz, Dz0 exchangeable), 6.30 (s, 2 H, NHz, D20 exchangeable), 10.78 (s, 1H, NH, DzOexchangeable), 12.06 (s, 1H, NH, D20 exchangeable); UV A, nm (< X 104) (MeOH-DMF, v:v, 8 2 ) 258 (1.3), 293 (0.8); (pH 1) 289 (0.9); (pH11) 257 (1.2), 288 (0.8). Anal. (CiiHisNeOd c , H, N. 8-Amino-9-(cyclohexylmethyl)guanine(9c). Compound 9c was prepared in 64% yield using a procedure similar to that which afforded 9a: mp >300 "C; UV A,, nm (e X lo4) (0.1 N NaOH) 253 (0.68); lH NMR (100 MHz, DMSO-ds) 6 0.94-1.63 (m, 11H), 3.58 (d, 2 H, CHz),5.79 (s,2 H, "2, DzO exchangeable), 6.12 (s, 2 H, NHz, D20 exchangeable), 10.30 (5, 1 H, NH, D20 exchangeable). Anal. (Cl~H1&0.~/4Hz0)C, H, N. 8-Amino-9-(naphthylmethyl)guanine (9d). Compound 9d was prepared in 67% yield using a procedure similar to that which afforded 9a: mp >300 "C; UV A, nm (e x lo4) (0.1 N NaOH) 282 (1.08); 'H NMR (100 MHz, D M s 0 - d ~6) 5.5 (s,2 H, D20 exchangeable), 6.14 (s, 2 H, NHz, CHz), 5.92 ( 8 , 2 H, "2, DzO exchangeable), 7.31-8.24 (m, 7 H, ArH), 10.4 (s, 1 H,NH, D20 exchangeable). Anal. (C~&~N&-HZO)C, H, N. 8-Amino-g-[m-( hydroxymethyl)phenyl]guanine (9e). Compound 9ewas prepared in 73% yield using a procedure similar nm (t X lo4) to that which afforded 9a: mp >300 "C: UV A, (0.1 N NaOH) 270 (1.2); lH NMR (100 MHz, DMSO-ds) 6 4.55 (d, 2 H, CHz), 5.28 (t,1H, OH, DzO exchangeable), 5.54 (s,2 H, "2, D20 exchangeable), 6.14 (s,2 H, "2, D20 exchangeable), 7.50-7.21 (m, 5 H, ArH), 10.39 (s, 1 H, NH, DzO exchangeable). Anal. (C1~H1~N602-H~0) C, H,N. 9-[(2-Iodoethoxy)methyl]guanine (16). A mixture of acyclovir (225 mg, l mmol), triphenylphosphine (262 mg, l mmol), and iodine (254 mg, 1 mmol) in anhydrous DMF (5 mL) was allowed to stand a t room temperature for 3 days. The solvent was then evaporated to dryness at 60 "C in vacuo (oil pump). The residue was purified by silica gel column chromatography using methanol-chloroform ( 2 5 ) as eluent. The proper portions of eluent were collected and evaporated to dryness to afford 9-[(2iodoethoxy)methyl]guanine(130 mg, 39%): mp 180 "C dec; UV A, nm (e X lo4) (methanol) 254 (1.5); (pH 1)256 (1.3); (pH 11) 264 (1.2); 'H NMR (270 MHz, DMSO-ds) 6 3.25 (t, 2 H, CHz), 3.71 (t, 2 H, CHz), 5.37 (9, 2 H, CHz), 6.50 (8, 2 H, "2, Dz0 exchangeable), 7.83 (s, 1 H, NH, DzO exchangeable), 10.62 (s, 1 H, NH, DzO exchangeable). Anal. (CsHloN5021) C, H, N. 8-Amino-9-[(2-iodoethoxy)methyl]guanine (17). A solution of 13 (0.3 g, 1.25 mmol), triphenylphosphine (0.33 g, 1.26 mmol) and iodine (0.32 g, 2.5 mmol) in anhydrous DMF (15 mL) was allowed to stand at room temperature for 24 h. Sodium dithionite (0.3 g) was added to the mixture and the mixture was then dissolved in a mixture of DMF and water (l:l,v:v, 20 mL). This solution was coevaporated with silica gel (2.5 g) to dryness a t 60 "C in vacuo. The solid was subjected to column chromatography (silica gel) and was eluted with chloroform-methanol (7:3, v:v). The proper fraction was collected and evaporated to dryness to furnish 8-amino-9-[(2-iodoethoxy)methyl]guanine(100 mg, nm (e X lo4) (methanol) 257 23%): mp 272-273 "C; UV A, (1.3), 293 (0.7); (pH 1)250 (1.3), 288 (0.8); (pH 11)259 (1.1);'H NMR (270 MHz, DMSO-ds) 6 3.30 (t, 2 H, CHz), 3.71 (t, 2 H, CH2),5.24 (s, 2 H, CHz),6.03 (s, 2 H, NHz, DzO exchangeable), 6.26 (s, 2 H, "2, DzO exchangeable), 10.53 (s, 1 H, NH, D20 exchangeable). Anal. (C~H~~N,@ZI) N. 9-[[2-[p-(Fluorosulfonyl)benzoyl]ethoxy]methyl]guanine (18). Acyclovir (1.0 g, 4.4 mmol) was coevaporated with DMF (2 X 10 mL) at 60 "C in vacuo and then was dissolved in HMPA (10 mL) with slight warming. The solution was immersed in an ice bath and p-(fluorosulfony1)benzoyl chloride (1.2 g, 5.4 mmol) was added into the mixture in small portions over a 3-h period. The mixture was stirred in an ice bath for another 1 h and was then poured into ice-water (100 mL) to afford a precipitate. The white solid was collected by filtration and recrystallized from methanol to furnish 9-[ [2-[p-(fluorosulfonyl)benzoyl]ethoxy]methyl]guanine (1.5 g, 82 %): mp 237238 "C; UV A, nm (e X lo4) (methanol) 255 (1.5); (pH 1) 257 (1.3); (pH 11) 265 (1.2); 'H NMR (270 MHz, DMSO-de) 6 3.38 (t,2 H, CHz), 4.41 (t,2 H, CHz), 5.39 ( 8 , 2 H, CHz), 6.48 (9, 2 H, NHz, DzO exchangeable), 7.84 (s, 1 H, H-8), 8.13 (d, 2 H, ArH, J = 8.4 Hz), 8.25 (d, 2 H, ArH, J = 8.4 Hz), 10.58 (s, 1 H, NH, D20 exchangeable). Anal. ( C ~ ~ H ~ ~ N ~ F S J /C, ZH H.Z O )

Journal of Medicinal Chemistry, 1993, Vol. 36, No. 8 1029

8-Amino-5'-deoxy-5'-(phenylthio)guanosine(19). Tributylphosphine (6.0 mL, 24.05 mmol) was added to a suspension of 8-aminoguanosineZ6(12, 2.08 g, 6.98 mmol) and diphenyl disulfide (3.5 g, 15.56 mmol) in pyridine (40 mL). The mixture was stirred at room temperature for 60 hand then poured slowly into a stirred mixture of water (100 mL) and diethyl ether (100 mL). The resulting solid was collected, washed with chloroform (10 mL) and diethyl ether (20 mL). The solid was recrystallized from a mixture of acetone and water (v:v, 1:l)to afford 8-amino5'-deoxy-5'-(phenylthio)guanosine (1.6g, 59%): mp 240-242 "C; 'H-NMR (360 MHz, DMSO-de) 6 3.4-3.28 (m, 2 H, H-57, 3.85 (9, 1 H, H-4'),4.15 (4, 1 H, H-3'1, 4.91 (9, 1 H, H-27, 5.12 (d, 1 H, H-3' OH, J = 5.4 Hz), 5.34 (d, 1 H, H-2' OH, J = 6.08 Hz), 5.58 (d, 1 H, H-1', J 5.76 Hz), 5.98 (s, 2 H, NHz, DzO exchangeable), 6.39 (s, 2 H, "2, DzO exchangeable), 7.14-7.35 (m,5H,ArH),11.04(s,l H,NH);UVA,,,nm(eX lo4)(methanol) 254 (2.3), 292 (0.83); (pH 1) 251 (2.1), 291 (0.87); (pH 11) 254 (2.0). Anal. ( C ~ ~ H I ~ N c~, SH,ON. ~) 8-Amino-5'-[p(fluorosulfonyl)benzoyl]guanosine (20). Concentrated hydrochloric acid (0.7 mL) was added to a stirred suspension of 8-aminoguanosineZ6(12, 1.0 g, 3.36 mmol) in anhydrous DMF (10 mL). The mixture was allowed to stir at room temperature. After 15min, THF (70 mL) was added to the mixture (clear solution) and the upper layer was decanted. Methanol (10 mL) was added to the yellowish oily residue and then anhydrous ether (50mL) afforded a precipitate. The white solid was collected by filtration and dried in vacuo (water pump) over PzO5 and silica gel for 12 h to furnish 8-aminoguanosine dihydrochloride (1.10 9). The dihydrochloride salt was then dissolved in HMPA (8 mL). The solution was immersed in an oil bath andp-(fluorosulfony1)benzoylchloride (0.72 g) was added in small portions to this solution. The reaction mixture was allowed to stir at room temperature for 48 h and was then extracted with petroleum ether (70 mL) and a mixture of ethyl acetate-ether (1:l)(100mL). The resulting yellowishoilyresidue was dissolved in methanol (10 mL). Ether (100 mL) was added to this solution to afford a precipitate. The white solid was collected by filtration and subsequently dissolved in anhydrous DMF (10 mL). Anhydrous triethylamine (1mL) was added to the resulting solution to afford a precipitate. To the suspension was added ether (100 mL) slowly with stirring. The light yellow solid was collected by filtration and air-dried. The yellow solid was recrystallized from a mixture of ethanol and water (1:2) (150 mL) with activated charcoal (1 g) to afford 8-amino-5'-[~(fluorosulfonyl)benzoyl]guanosine (0.6 g, 37%1: mp 240 "C dec; 'H-NMR (270 MHz, DMSO-de) 6 4.06 (q, 1 H, H-47, 4.56-4.45 (m, 2 H, H-5'),4.65 (q, 1H, H-3'1, 4.75 (q, 1 H, H-27, 5.07 (d, 1 H, H-3' OH,J = 6.3 Hz, DzO exchangeable), 5.42 (d, 1 H, H-2' OH, J = 5.4 Hz, DzO exchangeable), 5.63 (d, 1 H, H-1', J = 4.13 Hz), 5.86 (s,2 H, "2, DzO exchangeable), 6.17 (s,2 H, "2, DzO exchangeable), 8.24 (s, 4 H, ArH), 10.50 (s, 1 H, NH, DzO exchangeable); UV A,, nm (e X lo4) (methanol) 256 (1.9), 289 (1.1);(pH 1) 288 (1.2). Anal. (CIJ-I~~N,&QF) C, H, N. 5'-[p(Fluorosulfonyl)benzoyl]guanosine (21). Guanosine (1.0 g, 3.5 mmol) was coevaporated with anhydrous DMF (2 X 10 mL) at 60 OC in vacuo and then was dissolved in hexamethylphosphoric triamide (8 mL) with slight warming. The solution was immersed in an ice bath and to this solution was added p-(fluorosulfony1)benzoyl chloride (1.5 g, 6.7 mmol) in small portions. The mixture was allowed to stir a t room temperature for 4 h and was then extracted with petroleum ether (50 mL). The upper layer was discarded and the lower layer became a yellowish oil by the addition of ethyl acetate-ether (1:l)(100 mL). The resulting yellowish oil was further washed twice with ethyl acetate-ether (1:l)(50 mL). The resulting solid was crystallized from a mixture of methanol and water to afford 5'-[p-(fluorosulfonyl)benzoyl]guanosine (0.26g,26%): 'H-NMR (270 MHz, DMSO-de) 6 4.17 (q, 1 H, H-4'), 4.32 (t, 1 H, H-3'), 4.50 (m, 2 H, H-5'),4.61 (q, 1H, H-2'), 5.35 (br s, 1H, H-3' OH, DzOexchangeable), 5.59 (brs, 1H, H-2' OH, DzO exchangeable), 5.73 (d, 1 H, H-1', J = 4.7 Hz), 6.48 (5, 2 H, NHz, DzO exchangeable), 7.87 ( 8 , 1 H, =CH), 8.26 (e, 4 H, ArH), 10.64 (s, 1 H, NH, DzO exchangeable). 5'-Deoxy-5'-(phenylthio)formycin B (23). To a suspension of formycin B (1.0 g, 3.7 mmol) and diphenyl disulfide (1.84 g, 8.42 mmol) in pyridine (30 mL) was added tributylphosphine

1030 J o u r n a l of Medicinal Chemistry, 1993, Vol. 36, No. 8

(3.0 mL). The mixture was allowed to stir at room temperature for 3 h and then concentrated in vacuo at 60 “C to 10 mL. To the concentrated mixture was added ether (50mL), with shaking, to afford a white solid. The solid was collected by filtration and washed with ether (100 mL) to afford 1.0 g of the crude product. The filtrate was evaporated to dryness and then to this oil residue was added water (20 mL), methanol (5 mL), and ether (100 mL) to obtain another 0.4 g of crude product. The total crude product (1.4 g) was recrystallized from acetone and water to furnish 5’deoxy-5’-(phenylthio)formycin B (1.2 g, 89%): mp 207-209 “C; UV A, nm (c X lo4)(methanol) 255 (1.5); (pH 11)252 (1.2), 280 (0.9). lH-NMR (270 MHz, DMSO-&) 6 3.30 (m, 2 H, H-5’),3.92 (4, 1 H, H-49, 4.14 (9, 1 H, H-37, 4.56 (4, 1 H, H-29, 4.92 (d, 1 H, H-1’, J = 5.53 Hz), 5.09 (d, 1 H, H-2’ OH, J = 5.8 Hz, D2O exchangeable),7.30 (m, 5 H, ArH), 7.89 (s, 1 H, =CH), 12.28 (s, 1 H, NH, D20 exchangeable), 14.07 (s, 1 H, NH, D20 exchangeable). Anal. (C16H16N4O4S) C, H, N. 5’-[p(Fluorosulfonyl)benzoyl]formycin B (24). To a suspension of formycin B (0.5 g, 1.87 mmol) in HMPA (10 mL) was addedp-(fluorosulfony1)benzoylchloride (0.5g). The mixture was allowed to stir at room temperature for 12 h. The mixture was then poured into ice-water to furnish a white solid precipitate. The solid was collected by filtration and recrystallization from methanol to afford 5’-[p-(fluorosulfonyl)benzoyl]formycinB (0.6 g, 71%): mp 244-246 “C; ‘H-NMR (270 MHz, DMSO-&) 6 4.15 (t, 1 H, H-49, 4.61-4.24 (m, 4 H, H-5’, H-2’ and H-39, 5.04 (d, 1H, H-1’, J = 4.35 Hz), 5.15 (t,2 H, H-2’ OH, D20exchangeable), 7.81 (d, 1 H, =CH, J = 3.2 Hz), 8.30 (s, 4 H, ArH), 12.25 (s, 1 H, NH, DzO exchangeable), 14.12 (s,1H, NH, D20 exchangeable). Anal. (C17HlbN40&3FJ/4H20)C, H, N. PNP Inhibitory Assay.1s Substrate, [8-14C]inosine,is used at a specific activity of 22.5 mCi/mmol. For K , determinations, a fixed amount of diluted cell extract is incubated with variable amounts of radiolabeled substrate (12.5-100 mM) and inorganic phosphate (50 mM). Ki determinations were performed with variable radiolabled inosine concentrations (12.5-100 mM), fixed inorganic phosphate (50 rM) and variable inhibitor concentrations (0.03 mM to 1.0 pM). All reactions are incubated for 10 min at 37 “C. Substrate and product (inosine and hypoxanthine, respectively) are separated by high voltage paper electrophoresis. The radiolabeled product of the reaction is visualized by UV light (290 nm), cut from the paper, and counted in a toluenebased scintillation fluid in a Packard Tri-carb liquid scintillation spectrometer. Enzyme-free blank reactions are used as controls for all reactions. In all initial velocity determinations, with or without inhibitor, not more than 15% of the substrate is converted to product. Double reciprocal plots of the initial velocity values versus the substrate concentrations are linear.

Acknowledgment. This investigation was supported by research grants (No. NSC75-0412-B016-64and NSC760208-M016-01 to J.-W.C.) from National Science Council of the Republic of China and by the UNDP/World Bank/ WHO Special Programs for Research and Training in Tropical Diseases and Scientific Work Group of Filariasis (I.D. 840398 and 870387 to L.B.T.). References (1) Previous paper in this series: Chern, J.-W.; Lin, G.-S.; Chen, C.4.

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